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Research Administration and Compliance

Guidelines on Drop Method for Inhalation Anesthesia/Euthanasia in Rodents

This standard provides guidance in the proper use of a sealed container for rapid anesthetization of mice or rats and safety of the user. All researchers and animal handlers engaged in the use of isoflurane for short-term (usually 30-60 seconds) anesthesia or euthanasia of rodents, without the use of a vaporizer.

Standard

Inhalational anesthesia/euthanasia, also known as the ‘drop method’, is a rapid method to anesthetize rodents for very short procedures or to perform euthanasia via overdose.  For a sealed container, laboratories can purchase desiccators; either glass or plastic, from various vendors or the lab may construct a custom version for use.  Regardless of version, it is essential that the container be the appropriate size required to prevent overcrowding of animals and the need for large volumes of the isoflurane, which slows induction time. The container lid must be tight fitting to prevent potential leaks of isoflurane and is easily sanitizable. If possible, a slide-top lid should be used over a hinged lid to prevent isoflurane gas from being pushed into the user’s breathing zone. If this is impossible, then facing the hinge opening of the lid away from the user is recommended. 

A protective layer (such as a perforated platform in a desiccator or a small tissue cassette or other small device to hold the material) is required inside the container to prevent direct contact of the animals with isoflurane soaked materials. Contact with the anesthetic can cause skin irritation and potential overdose since isoflurane is absorbed through the skin.

Due to potential risk of human health, the use of the ‘drop method’ must occur in a fume hood, a ducted biosafety cabinet, or with a properly functioning active scavenging system.  If this cannot occur, EHS approval is required to use the ‘drop method’ on a bench top.  An isoflurane exposure monitoring must be performed by EHS to protect personnel from inhalation exposure to anesthetic gases. Please contact EHS at 919-515-6655 or email env-health-occ-health@ncsu.edu. Once approved, a posted copy of the approval (specific for such procedure) in the area is required.

To be performed inside a chemical fume hood or hard ducted biosafety cabinet, such as Class II, Type B2 BSC:

  1. Pre-charge the sealed container by placing the isoflurane-soaked material.  Close the lid and wait 5 minutes for the liquid to form a volatile gas within the chamber. (Note: the amount of isoflurane to add depends on the size of the chamber, so the actual amount needed may vary.)
  2. Remove the lid of the container, quickly place the animals onto the protective layer within the chamber, and immediately close the lid. (For perforated platforms, ensure that the perforations are not large enough for the animal to crawl through and risk contact with the isoflurane soaked material.)
  3. The animals will first exhibit an excitatory phase, after which they become anesthetized. Monitor animal closely. A deep plane of anesthesia is indicated by lack of a righting reflex when the jar is tipped slightly and a respiratory rate that is reduced by ~50% from pre-anesthetic rate (i.e. 80-100 breaths/min).  This should take ~1 min for mice and ~2 min for rats. Observe the animal closely so that overdose does not occur. (Mouse or Rat Neonates up to 10 days of age must remain in the chamber for at least five (5) minutes)
  4. Allow animal to remain in deep anesthesia for ~10 sec before proceeding.  Remove animal from the anesthesia container and replace lid immediately.
  5. To ensure an adequate plane of anesthesia, apply a noxious stimulus (i.e. toe pinch) before proceeding with the procedure.  If the animal responds to the toe pinch, return it to the anesthesia container. An absent withdrawal reaction indicates appropriate anesthetic depth and the procedure can be performed.
Note: Isoflurane is highly volatile and animals will quickly regain consciousness once removed from the chamber; thus, it is imperative to perform the procedure immediately. A nosecone can be used for longer procedures

Nose Cone 

position nose cone just on the tip of the muzzle
  • Slightly moisten the end of a small piece of gauze with the Isoflurane. Insert the gauze into conical tube or syringe nose cone of appropriate size, with moistened end away from open end of the nose cone. Excess liquid anesthetic must not come in contact with the rodent.
  • Place the animal’s muzzle at the edge of the nose cone. Moisten the animal’s eyes with Paralube. Check the depth of anesthesia as described as step #5 above (the depth of anesthesia can be adjusted by moving the animal’s nostrils closer to or further from the end of the cone). Care must be taken not to create a complete seal around the muzzle.
    • Once the recovery procedure is completed, place the animal in a clean cage, bedding covered if present, until the mouse/rat is fully ambulatory. 
    • If utilizing the drop method for euthanasia, the animal should be left inside the container until cessation of breathing is observed (Mouse or Rat Neonates up to 10 days of age may require longer exposure).  Remove the animal quickly and perform a secondary physical method.
Note: All gas euthanasia must be followed with a secondary physical method.

References

Revised 12/2024